1. Field of the Invention
The invention relates to mammalian kinase ligands, substrates and modulators. In particular, the invention relates to polypeptides, polypeptide compositions and polynucleotides that encode polypeptides that are ligands, substrates, and/or modulators of PKC. The invention also relates to polyligands that are homopolyligands or heteropolyligands that modulate PKC activity. The invention also relates to ligands and polyligands tethered to a subcellular location.
2. Background of the Invention
Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian Protein Kinase C is also known as PKC. PKC can phosphorylate serine and threonine residues in protein or peptide substrates. The enzymatic activity, activation and regulation of PKC have been studied. Many cellular substrates of PKC have been identified (See for example, Ahmed et al. 2005 FEBS J 272:903-915; Anthonsen et al. 2001 J Biol Chem 276:35344-51; Behn-Krappa et al. 1999 Curent Biology 9:7298-37; Burchfield et al. 2004 J Biol Chem 279:18623-32; Buther et al. Biochem Biophys Res Commun 2004:703-707; Cenni et al. 2002 Biochem J 363:537-45; Edlund et al. 1998 FEBS Letters 425:166-70; Eichholtz et al. 1993 J Biol Chem 268:1982-86; Ho et al. 1997 J Neurochem 68:2514-22; Jain et al. 1999 J Biol Chem 274:24392-24400; Jayanthi et al. J Biol Chem Jun. 1, 2006 manuscript M601156200; Jayanthi et al. 2006 J Biol Chem 281:23326-40; Johnson et al. 1996 J Biol Chem 271:24962-66; Koponen et al. 2003 J Neurochem 86:442-50; Li et al. 2004 EMBO J 23:1112-1122; Liu et al. 2002 Biochem J 361:255-265; Nika et al. 2006 Mol Cell Biol 26:1806-1816; Nishikawa et al. 1997 J Biol Chem 272:952-60; Noland et al. 1989 J Biol Chem 264:20778-785; Oehrlein et al. 1996 Biochem J 317:219-224; Pietromonaco et al. 1998 J Biol Chem 273:7594-603; Ren et al. 2002 J Biol Chem 277:33758-765; Rodriguez et al. 1999 FEBS Letters 454:240-46; Ron et al. 1995 J Biol Chem 270:24180-87; Satoh et al. 2004 Am J Physiol Gastrointest Liver Physiol 287:G582-G591; Shimazaki et al. 1996 J Biol Chem 271:14548-53; Sommerfeld et al. 2004 Biochemistry 43:5888-5901; Song et al. 2006 J Biol Chem 281:15582-91; Strack et al. 2002 Biochemistery 41:603-608; Thuille et al. 2005 EMBO J 24:3869-80; Uddin et al. 2002 J Biol Chem 277:14408-14416; Verghese et al. 1994 J Biol Chem 269:9361-67; Walaas et al. 1994 Biochem J 302:635-40; Ward et al. 1993 Biochemistry 32:11903-11909; Watson et al. 1999 Biochem J 343:301-305; Yang et al. 2005 J Biol Chem 280:207-214; Zhang et al. 2005 J Neurosci 25:8468-81).
There are several small molecule agents known in the art and used experimentally, such as staurosporine, K-252a, UCN-01, tamoxifen, benzolactams, ruboxistaurin that modulate PKC activity. Natural and synthetic polypeptides have been studied to examine PKC substrate specificity. While polypeptides and variants thereof have been studied as individual PKC substrates or ligands, mixed ligands linked together as polyligands that modulate PKC activity have not been demonstrated before this invention. An aspect of the invention is to provide novel, modular, inhibitors of PKC activity by modifying one or more natural substrates either by truncation or by amino acid substitution. A further aspect of the invention is the subcellular localization of a PKC inhibitor, ligand, or polyligand by linking to a subcellular localization signal.
Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also U.S. Pat. No. 7,071,295.